Functional Analysis of Blood Pressure Genome Wide Association Study Loci

Blood pressure (BP) is a highly heritable trait influenced by the complex interaction between environmental and multiple genetic components. The heritability of BP is calculated to be 30–50%, and genome wide association studies (GWAS) have identified a large number of BP associated genetic markers. However, the majority of these fall in non-coding regions, and it is difficult to pinpoint the genes they affect. Non-coding genetic variants can have an effect on the transcription of causal genes through altering the efficiency of their regulatory elements (REs) and it is well established, that REs, enhancers and silencers, act on their target genes by forming a chromatin loop with the gene promoter. Therefore, we hypothesized that some intergenic BP associated variants are located in REs that form chromatin loops with genes that are linked to BP and affect their expression levels. We used publicly available data on BP associated variants and chromatin conformation data in tissues relevant for BP to identify candidate variant-gene pairs, where the common intergenic variant likely affects the expression of the target gene through the altered long-range regulatory interaction. We identified 7 candidate pairs and selected two for validation in human umbilical vein endothelial cells (HUVECs) to test the effect of the variant on the expression of its putative target gene. We used the CRISPR-Cas9 system to introduce the variants and we compare the expression level of the target gene in cells carrying the alternative and reference alleles. Our study explores the distal effects of intergenic single nucleotide polymorphisms (SNPs) and once confirmed, it will underline the complexity of the observed GWAS signals and demonstrate that there might not be a single causal gene in each locus.